Studying the role of clonal hematopoiesis in mouse solid tumors using a native-tissue relevant context model

• Project/Area Number: 21K15478
• Research Category: Grant-in-Aid for Early-Career Scientists
• Allocation Type: Multi-year Fund
• Review Section: Basic Section 50010:Tumor biology-related
• Research Institution: University of Tsukuba
• Principal Investigator: NGUYEN BICH・TRAN 筑波大学, 医学医療系, 研究員 (10812901)
• Project Period (FY): 2021-04-01 – 2024-03-31
• Project Status: Granted (Fiscal Year 2022)
• Budget Amount: ¥4,680,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥1,080,000); Fiscal Year 2023: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000); Fiscal Year 2022: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000); Fiscal Year 2021: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000)
• Keywords: Clonal hematopoiesis / TET2 / Colon cancer / Clonal Hematopoiesis / Tet2 / Melanoma

Outline of Research at the Start

Clonal hematopoiesis (CH) with adverse survival outcomes has been found in 24% of solid cancer patients. TET2 and SRSF2 mutations are commonly found in CH. We made a colon tumor model in the livers of Tet2KO and Srsf2 mutated mice to study the effect of CH immune cells on tumor cells and the cancer cell stage susceptible to these cells in a native tissue-relevant context.

Outline of Annual Research Achievements

Backgrounds: Immune cells with somatic mutations (IMsm) are infiltrated into cancer tissues in cancer patients with clonal hematopoiesis (CH). The roles of IMsm may vary depending on cancer types. Aim: To examine the roles of IMsm in colon cancer metastasis.
Methods: We transplanted colon cancer organoid cells into spleens of various Tet2 conditional knockout mice: VAV1Cre (Tet2 gene deletion in all hematopoietic cells), LysMCre (myeloid), CD19Cre (B), and CD4Cre (T). Livers were collected 30 days after transplantation. Liver metastasis tumor burden (LMTB) was defined by numbers of tumor foci, manually counted in 10 slides. Sorted CD4+, CD8+, CD11b+, and CD19+ cells were subjected to whole transcriptome analysis (WTA), respectively.
Results: LMTB of VAV1Cre and CD4Cre were lower than those of control (VAV vs cont, 55 vs 77 foci/1000 mm2; p<0.05; CD4 vs cont, 55 vs 86 foci/1000mm2; p<0.05), while that of CD19Cre and LysMCre were comparable. Differentially expressed gene (DEG) analysis for WTA showed that Pdcd1, Tim3, Tigit, and Lag3, encoding inhibitory receptors were repressed in CD8+ cells sorted from VAV1Cre and CD4Cre livers comparing to those of control. Immunofluorescence staining showed that PDCD1+TIM3+CD8+ cells were decreased in VAV1Cre livers comparing to control. We also found that transcription factor X that regulates the expression of the inhibitory receptors was highly expressed in tumor infiltrated-CD8 cells of control mice compared those of VAV1Cre and CD4Cre mice.

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

We finished 4 out of all 5 research plans. Our research showed that deficiency of Tet2 gene in hematopoietic cells led to decrease of exhausted CD8+ T cells and suppressed the liver metastasis of colon cancer organoid cells.

Strategy for Future Research Activity

1) Plan 1: Inhibit exhausted T cell by anti PD1 antibody to prove the role of the exhausted T cells in liver colon cancer metastasis. Control and VAV1Cre mice will be treated with anti PD1 antibody or istotype. Liver metastasis tumor burden will be evaluated.
2) Plan 2: Demonstrate relationship of the transcription X and Tet2 in CD8-T cells
-Plan 1.1: Check methylation status of the transcription X in CD8-T cells of control, VAV1Cre and CD4Cre mice. Methylation status will be checked by using MethylCollector MBD capture kit (Active Motif).
-Plan 1.2: Check methylation status of the transcription X after treated with a de-methylation drug in CD8-T cells VAV1Cre mice. Methylation status will be checked by using MethylCollector MBD capture kit (Active Motif).
3) Plan 3: Perform treatment experiment with a Tet inhibitor. Control mice will be treated with the Tet inhibitor or vehicle. Liver metastasis tumor burden will be evaluated.

Research Products

• [Journal Article] Clonal germinal center B cells function as a niche for T-cell lymphoma (2022)
  • Author(s): Fujisawa M, Nguyen TB, Abe Y , Suehara Y, Fukumoto K, Suma S, Makashima K, Kaneko C, Nguyen YTM, Usuki K, Narita K, Matsue K, Nakamura N, Ishikawa S, Miura F, Ito T, Suzuki A, Suzuki Y, Mizuno S, Takahashi S, Chiba S, and Sakata-Yanigimoto M
  • Journal Title: Blood Volume: 140 (18) Issue: 18 Pages: 1937-1950
  • DOI: 10.1182/blood.2022015451
  • Peer Reviewed / Open Access / Int'l Joint Research